Carboxypeptidase of cocoa

ABSTRACT

The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase and polypeptide in the manufacture of cocoa flavor and/or chocolate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of the US national phasedesignation of International application PCT/EP02/07162 filed Jun. 28,2002, the content of which is expressly incorporated herein by referencethereto.

BACKGROUND ART

The present invention relates to a novel carboxypeptidase gene and thepolypeptide encoded thereby. In particular, the present inventionrelates to the use of the present carboxypeptidase in the manufacture ofcocoa flavor and/or chocolate.

It is known that in processing cacao beans the generation of the typicalcocoa flavor requires two steps—a fermentation step, which includesair-drying of the fermented material, and a roasting step.

During fermentation two major activities may be observed. First, thepulp surrounding the beans is degraded by micro-organisms with thesugars contained in the pulp being largely transformed to acids,especially acetic acid (Quesnel et al., J. Sci. Food. Agric. 16 (1965),441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). Theacids then slowly diffuse into the beans and eventually cause anacidification of the cellular material. Second, fermentation alsoresults in a release of peptides exhibiting differing sizes and ageneration of a high level of hydrophobic free amino acids. This latterfinding led to the hypothesis that proteolysis occurring during thefermentation step is not due to a random protein hydrolysis but seems tobe rather based on the activity of specific endoproteinases (Kirchhoffet al., Food Chem 31 (1989), 295-311). This specific mixture of peptidesand hydrophobic amino acids is deemed to represent cocoa-specific flavorprecursors.

Until now several proteolytic enzyme activities have been investigatedin cacao beans and studied for their putative role in the generation ofcocoa flavor precursors during fermentation.

An aspartic endoproteinase activity, which is optimal at a very low pH(pH 3.5) and inhibited by pepstatin A has been identified. A polypeptidedescribed to have this activity has been isolated and is described toconsist of two peptides (29 and 13 kDa) which are deemed to be derivedby self-digestion from a 42 kDa pro-peptide (Voigt et al., J. PlantPhysiol. 145 (1995), 299-307). The enzyme cleaves protein substratesbetween hydrophobic amino acid residues to produce oligopeptides withhydrophobic amino acid residues at the ends (Voigt et al., Food Chem. 49(1994), 173-180). The enzyme accumulates with the vicilin-class (7S)globulin during bean ripening. Its activity remains constant during thefirst days of germination and does not decrease before the onset ofglobulin degradation (Voigt et al., J. Plant Physiol. 145 (1995),299-307).

Also a cysteine endoproteinase activity had been isolated which isoptimal at a pH of about 5. This enzymatic activity is believed not tosplit native storage proteins in ungerminated seeds. Cysteineendoproteinase activity increases during the germination process whendegradation of globular storage protein occurs. To date, no significantrole for this enzyme in the generation of cocoa flavor has been reported(Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, 17-23Nov. 1996).

Moreover, a carboxypeptidase activity has been identified which isinhibited by PMSF and thus belongs to the class of serine proteases. Itis stable over a broad pH range with a maximum activity at pH 5.8. Thisenzyme does not degrade native proteins but preferentially splitshydrophobic amino acids from the carboxy-terminus of peptides (Bytof etal., Food Chem. 54 (1995), 15-21).

During the second step of cocoa flavor production—the roasting step—theoligopeptides and amino acids generated at the stage of fermentation areobviously subjected to a Maillard reaction with reducing sugars presentin fermented beans eventually yielding substances responsible for thecocoa flavor as such.

In the art there have been many attempts to artificially produce cocoaflavor.

Cocoa-specific aroma has been obtained in experiments wherein acetonedry powder (AcDP) prepared from unfermented ripe cacao beans wassubjected to autolysis at a pH of 5.2 followed by roasting in thepresence of reducing sugars. It was conceived that under theseconditions preferentially free hydrophobic amino acids and hydrophilicpeptides should be generated and the peptide pattern thus obtained wasfound to be similar to that of extracts from fermented cacao beans. Ananalysis of free amino acids revealed that Leu, Ala, Phe and Val werethe predominant amino acids liberated in fermented beans or autolysis(Voigt et al., Food Chem. 49 (1994), 173-180). In contrast to thesefindings, no cocoa-specific aroma could be detected when AcDP wassubjected to autolysis at a pH of as low as 3.5 (optimum pH for theaspartic endoproteinase). Only few free amino acids were found to bereleased but a large number of hydrophobic peptides were formed. Whenpeptides obtained after the autolysis of AcDP at a pH of 3.5 weretreated with carboxypeptidase A from porcine pancreas at pH 7.5,hydrophobic amino acids were preferentially released. The pattern offree amino acids and peptides was similar to that found in fermentedcacao beans and to the proteolysis products obtained by autolysis ofAcDP at pH 5.2. After roasting of the amino acids and peptides mixtureas above, a cocoa aroma could be generated.

It has also been shown that, a synthetic mixture of free amino acidsalone with a similar composition to that of the spectrum found infermented beans, was incapable of generating cocoa aroma after roasting,indicating that both the peptides and the amino acids are important forthis purpose (Voigt et al., Food Chem. 49 (1994), 173-180.

In view of the above data a hypothetical model for the generation,during fermentation, of the said mixture of peptides and amino acids,i.e. the cocoa flavor precursors, had been devised (FIG. 1), where in afirst step peptides having a hydrophobic amino acid at their end, areformed from storage proteins, which peptides are then further degradedto smaller peptides and free amino acids. To produce the said peptideshaving C-terminal hydrophobic amino acids, an aspartic endoproteinaseactivity related to that mentioned above seems to be involved. Yet, forsplitting off hydrophobic amino acids from peptides formed in thepreceding step the only known enzymatic activity, which might beconsidered in this respect, is that of a carboxypeptidase. However, suchenzyme has not been isolated and studied in detail in cacao and it istherefore still questionable, which cacao enzyme might be responsiblefor the generation of hydrophobic amino acids required for cocoa flavor.

Though some aspects of cocoa flavor production have been elucidated sofar there is still a need in the art to fully understand the processesinvolved, so that the manufacture of cocoa flavor may eventually beoptimized.

SUMMARY OF THE INVENTION

The present invention provides means for further elucidating theprocesses involved in the formation of cocoa-specific aroma precursorsduring the fermentation of cacao seeds, to improve the formation ofcocoa flavor during processing and manufacturing and eventuallyproviding means assisting in the artificial production of cocoa flavor.

This problem has been solved by providing a nucleotide sequence encodinga novel carboxypeptidase from cacao beans (termed cacao CP-III), whichis identified by SEQ. ID. No. 1, or functional derivatives thereofhaving a degree of homology that is greater than 80%, preferably greaterthan 90% and more preferably greater than 95%.

It will be appreciated by the skilled person that a gene encoding aspecific polypeptide may differ from a given sequence according to theWobble hypothesis, in that nucleotides are exchanged that do not lead toan alteration in the amino acid sequence. Yet, according to the presentinvention also nucleotide sequences shall be embraced, which exhibit anucleotide exchange leading to an alteration of the amino acid sequence,such that the functionality of the resulting polypeptide is notessentially disturbed.

This nucleotide sequence may be used to synthesise a correspondingpolypeptide by means of recombinant gene technology, in particular apolypeptide as identified by SEQ. ID. No. 2.

As has been shown in a comparison with other carboxypeptidases fromother plants the present enzyme does not show a substantial homology toany of the carboxypeptidases known so far. Since it is assumed, thatcocoa may furthermore contain additional carboxypeptidases that mightexhibit a higher homology to the carboxypeptidases known so far it mustbe considered as a surprising fact that this very enzyme has beendetected.

For producing the polypeptide by recombinant means, the nucleotide ofthe present invention is included in an expression vector downstream ofa suitable promoter and is subsequently incorporated into a suitablecell, which may be cultured to yield the polypeptide of interest.Suitable cells for expressing the present polypeptide include bacterialcells, such as e.g. E. Coli, or yeast, insect, mammalian or plant cells.

The present DNA sequence may also be incorporated directly into thegenome of the corresponding cell by techniques well known in the art,such as e.g. homologous recombination. Proceeding accordingly willprovide a higher stability of the system and may include integration ofa number of said DNA-sequences into a cell's genome.

The cells thus obtained may in consequence be utilized to produce thepolypeptide in batch culture or using continuous procedures, with theresulting polypeptide being isolated according to conventional methods.

The recombinant carboxypeptidase obtained may be used for themanufacture of cocoa flavor. To this end, the enzyme described hereinmay be utilized in an artificial trial run, wherein a mixture ofdifferent proteins, such as cacao storage proteins, or proteinhydrolysates of other resources, are subjected to enzymatic degradationby means of enzymes, known to be involved in proteolytic degradation toeventually assist in the production of flavor precursors. The enzyme maylikewise also be utilized in the production of cocoa liquor, and in themanufacture of chocolate.

Yet, the present invention also provides plants, in particular cacaoplants, comprising a recombinant cell, containing one or more additionalcopies of the carboxypeptidase of the present invention. Such a cacaoplant will produce beans, which will exhibit a modified degradation ofstorage proteins when subjected to the fermentation process, allowing amore rapid degradation or a pattern of hydrolysis that yields a higherlevel of cocoa flavor precursor, since a higher amount ofcarboxypeptidase will be present.

The carboxypeptidase of the present invention may also be used toproduce other transgenic plants such as soybean and rice, producingseeds with this new protein modifying enzyme.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

In the figures,

FIG. 1 shows a scheme illustrating a potential process for theproteolytic formation of cocoa-specific aroma;

FIG. 2 shows the cloning strategy used for the isolation of a cDNAencoding a carboxypeptidase from Theobroma cacao;

FIG. 3 shows a comparison of the hydrophilicity Plot-Kyte-Doolittle forthe cacao CP-III sequence with Barley CP-MI, CP-MII and CP-MIII;

FIG. 4 shows a Northern blot analysis of cacao CP-III.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As described above, it was suggested that a carboxypeptidase could beinvolved in the production of cocoa flavor precursors during cacaofermentation. However it was not known in the art which cacaocarboxypeptidase carried out this function considering that five classesof carboxypeptidases (Type I-V) have been identified in different plantsby references to differences in substrate specificities, molecularweights and chromatographic profiles. Furthermore 50 sequences havinghomologies with serine carboxypeptidases exist in the completedArabidopsis genome.

The following examples illustrate the invention further without limitingit thereto. In the examples the following abbreviations have been used:PCR: Polymerase Chain Reaction RACE: Rapid Amplification cDNA Ends cDNA:complementary deoxyribonucleic acid mRNA: messenger ribonucleic acidDEPC: Diethyl pyrocarbonate 3,4-DCI: 3,4-dichloroisocoumarin

EXAMPLES Materials

Cacao (Theobroma cacao L.) seeds (male parent unknown) from ripe pods ofclone ICS 95 were provided by Nestlé ex-R&D Center Quito (Ecuador). Theseeds were taken from the pods immediately after arrival at NestléResearch Center Tours (4-5 days after harvesting). The pulp and the seedcoat were eliminated and the cotyledons were frozen in liquid nitrogenand stored at −80° C. until use.

Preparation of mRNA

Total RNA was prepared using the following method. Two seeds were groundin liquid nitrogen to a fine powder and extraction was directlyperformed with a lysis buffer containing 25 mM Tris HCl pH8, 25 mM EDTA,75 mM NaCl, 1% SDS and 1M β-mercaptoethanol. RNA was extracted with onevolume of phenol/chloroform/isoamylalcohol (25/24/1) and centrifuged at8000 rpm, 10 min at 4° C. The aqueous phase was extracted a second timewith one volume of phenol/chloroform/isoamylalcohol (25/24/1). RNA wasprecipitated with 2M lithium chloride at 4° C. overnight. The RNA pelletobtained after centrifugation was resuspended in DEPC treated water anda second precipitation with 3M sodium acetate pH 5.2 was performed inpresence of two volumes of ethanol. The RNA pellet was washed with 70%ethanol and resuspended in DEPC treated water. Total RNA was furtherpurified using the Rneasy Mini kit from Qiagen®.

Cloning of a Carboxypeptidase cDNA

Cloning Strategy

A 1.5 kb 5′ end fragment of a carboxypeptidase from cacao seed wasamplified by RT-PCR using a degenerate oligonucleotide. Based on thesequence of this fragment, a primer was designed to amplify a 3′-endfragment. Finally, a full-length cDNA (cacao CP-III) was amplified usingprimers specific to both extremities (FIG. 2).

Primer Design

A search for carboxypeptidase sequences in the GenBank database lead tothe identification of several plant sequences. A multiple alignment ofthese sequences revealed the presence of conserved regions. Theconserved sequence MVPMDQP located near the histidine catalytic site hasbeen used to design a degenerate oligonucleotide in the antisenseorientation: pCP2r (5′-GGYTGRTCCATNGGNACCAT) (SEQ ID No. 3).

Synthesis of cDNA

Total RNA (see above) was used to synthesise first strand 3′ and 5′cDNAs with the SMART® RACE cDNA Amplification Kit (Clontech, USA).Synthesis has been performed exactly as described in the kitinstructions using 1 μg of total RNA and the Superscript™ II MMLVreverse transcriptase (Gibco BRL, USA). After synthesis, cDNAs were useddirectly for PCR or kept at −20° C.

5′ RACE Amplification

Specific cDNA amplification was performed with 2.5 μl of the firststrand 5′ cDNA in 50 μl buffer containing: 40 mM Tricine-KOH pH 8.7, 15mM KOAc, 3.5 mMMg(OAc)₂, 3.75 μg/ml BSA, 0.005% Tween-20, 0.005%Noninet-P40, 0.2 mM dNTP's, 14 pmoles of pCP2r primer, 5 μl of 10×Universal primer Mix (UPM) and 1 μl 50× Advantage 2 polymerase Mix(Clontech, USA). Amplification was performed in a Bio-med thermocycler60 (B. Braun). A first denaturation step (94° C., 2 min) was followed by35 cycles of denaturation (94° C., 1 min), primer annealing (55° C., 1.5min) and extension (72° C., 2 min). The extension time was increased by3 sec at each cycle. Amplification was ended by a final extension step(72° C., 10 min). The amplified fragment was cloned in pGEM®-T vectorand sequenced.

3′ RACE PCR

The sequence information obtained after the sequencing of the 5′ endfragment was used to design a specific oligonucleotide pCP5(5′-GCTTTTGCTGCCCGAGTCCACC) (SEQ ID No. 4), which was used for 3′-RACEamplification. 3′-RACE PCR was performed with 2.5 μl of SMART singlestrand 3′ cDNA in 50 μl buffer containing 40 mM Tricine-KOH pH 8.7, 15mM KOAc, 3.5 mM Mg(OAc)₂, 3.75 μg/ml BSA, 0.005% Tween-20, 0.005%Nonidet-P40, 0.2 mM dNTP's, 10 pmoles of pCP5 primer, 10 μl of 10×Universal primer Mix (UPM) and 1 μl 50× Advantage 2 polymerase Mix(Clontech, USA). Amplification was performed via touchdown PCR, in aBio-med thermocycler 60 (B. Braun).

A first denaturation step (94° C., 1 min) was followed by:

-   -   5 cycles including denaturation at 94° C. for 30 sec and        annealing/extension at 72° C. for 3 min    -   5 cycles including denaturation at 94° C. for 30 sec and        annealing/extension at 70° C. for 30 sec and 72° C. for 3 min    -   30 cycles including denaturation at 94° C. for 30 sec and        annealing/extension at 68° C. for 30 sec and 72° C. for 3 min.        The amplified fragment was cloned in pGEM®-T vector and        sequenced.        Full Length cDNA

The sequence information obtained after the sequencing of 5′- and3′-RACE fragments was used to design two specific oligonucleotides. (SEQID No. 5) pCP8: A sense primer (5′-CAAAGAGAAAAAGAAAAGATGGC) (SEQ ID No.6) pCP7r: A reverse primer (5′-CCCCAGAGCTTTACGATACGG).

PCR reaction was performed with 2.5 μl first strand cDNA in 50 μl buffercontaining: 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg(OAc)₂, 3.75μg/ml BSA, 0.005% Tween-20, 0.005% Noninet-P40, 0.2 mM dNTP's, 10 pmolesof pCP8 primer, 10 pmoles of pCP7r primer and 1 μl 50× Advantage 2polymerase Mix (Clontech, USA). Amplification was performed in a Bio-medthermocycler 60 (B. Braun). A first denaturation step (94° C., 1 min)was followed by 35 cycles of denaturation (94° C., 30 sec), primerannealing (63° C., 1 min) and extension (72° C., 2 min). The extensiontime was increased by 3 sec at each cycle. Amplification was ended by afinal extension step (72° C., 10 min). The amplified fragment was clonedin pGEM®-T Easy vector and sequenced.

Sequencing and Analysis of DNA Sequences

cDNA sequencing has been performed by Eurogentech (Belgium) and ESGS(France). Sequence analysis and comparison were performed with Lion'ssoftware bioScout, Lasergene software (DNAStar) and Genedoc programme.

The cacao CP-III cDNA sequence is 1768 bp long. A putative initiationstart codon was assigned by comparison with other carboxypeptidasesequences. It is located 25 bp from the 5′ end. The open reading frameis broken by a stop codon (TGA) at position 1549, followed by a putativepolyadenylation signal (TATAAA) at position 1725.

Cacao CP-III encodes a 508 amino acid type III carboxypeptidase C with apredicted molecular weight of 56 kDa and a pI of 5.04. The catalyticamino acids are present at position Ser²³⁸, Asp⁴¹⁶ and His⁴⁷³.Hydrophilicity analysis (FIG. 3) reveals that cacao CP-III encodes ahydrophilic protein with a very hydrophobic N-terminal end, indicatingthe presence of a signal peptide.

Northern Blot Analysis

Total RNA samples were separated on 1.5% agarose gel containing 6%formaldehyde (FIG. 4). After electrophoresis, RNA was blotted onto nylonmembranes (Appligene) and hybridized with ³²P-labeled cacao CP-III probeat 65° C. in 250 mM Na-phosphate buffer pH 7.2, 6.6% SDS, 1 mM EDTA and1% BSA. Cacao CP-III cDNA fragment was amplified by PCR using pCP8 andpCP7R primers and labelled by the random priming procedure (Rediprime®II, Amersham Pharmacia Biotech). Membranes were washed three times at65° C. for 30 min in 2×SSC, 0.1% SDS, in 1×SSC, 0.1% SDS and in 0.5×SSC,0.1% SDS.

1. (canceled)
 2. A polypeptide encoded by the nucleotide sequence codingfor a carboxypeptidase and having a sequence as identified by SEQ. ID.No. 1 or functional variants thereof having a degree of homology of morethan 90%. 3-12. (canceled)
 13. A method for hydrolysing proteins whichcomprises obtaining a polypeptide of claim 2 and utilizing thepolypeptide to hydrolyse the proteins.
 14. The method of claim 13wherein the proteins are derived from food material. 15-17. (canceled)18. A cocoa flavor produced by the method of claim
 2. 19. A compositioncomprising a food or beverage product and an effective amount of thecocoa flavor of claim 2.